The red cell membrane skeleton is a hexagonal protein lattice composed principally of spectrin and actin, which is attached to the overlying lipid bilayer through interactions of spectrin with ankyrin and band 3. Plasma membrane skeletons also exist in non-erythroid cells and there is increasing evidence that they will function in internal cellular structures such as the Golgi, lysosomes nucleus and vesicular transport systems. We will investigate three red cell membrane questions. Aim 1 will focus on the function of ankyrin regulatory domain and the redundancy of the ankyrins. We will complete an ankyrin 1 knockout and in vivo replacement of anykyrin 1 with ankyrin lacking a "regulatory" domain, or with full-length ankyrin 3. We will carefully measure the phenotype of the resulting mice and the effects of these changes in ankyrin on the structure of the membrane skeleton, the mobility of band 3, and the physical properties of the red cell membrane. Since removal of the regulatory domain will remove a conserved ankyrin "death domain", we will also look for apoptotic or anti-apoptotic effect on erythroblasts. Aim 2 is based on the observations that band 3, the red cell anion transporter, concentrates at the poles of dividing erythroblasts, and that zebrafish with the retsina allele, who lack band 3, have increased numbers of binucleate erythroblasts. This suggests that band 3 is involved in erythroblast cytokinesis. We will look for interaction of band 3 with spindle components or other proteins, using "pull-down" assays and two-hybrid screens, and we will also identify milder forms of the zebrafish retsina defect and use these fish in enhancer and suppressor screens for genes that modify the onset or degree of anemia. Such genes will be candidates for proteins that interact with band 3 in cytokinesis. Finally, in Aim 3, we will characterize bIII spectrin, which is located in the Golgi and in cytoplasmic vesicles. We will identify the vesicles, locate the vesicle binding site on spectrin and the spectrin-binding partner on the vesicles, and vesicles, and investigate the consequences of interfering with these interactions. Particular attention will be paid to the possibility that bIII spectrin is involved in ER-to-Golgi trafficking. We will also disrupt the spectrin bIII gene to assess the loss of function phenotype and to isolate fibroblast and hematopoietic cell lines lacking spectrin bIII for rescue experiments and other tests of bIII spectrin function. These experiments will broaden our knowledge of the membrane skeleton and help us begin to understand the diverse functions of this important cellular structure.